DNA for encoding D-hydantoin hydrolases, DNA for encoding N-carbamyl-D-amino acid hydrolases, recombinant DNA containing the genes, cells transformed with the recombinant DNA, methods for producing proteins utilizing the transformed cells and methods for producing D-amino acids

ABSTRACT

DNA for encoding a protein having D-hydantoinase activity which has a base sequence represented by Sequence ID No. 1 in the Sequence Listing. DNA for encoding a protein having D-carbamylase activity which has a base sequence represented by Sequence ID No. 3 in the Sequence Listing.

FIELD OF THE INVENTION

The present invention relates to a DNA for encoding D-hydantoinhydrolase (“D-hydantoinase”) which is utilized preferably for producingD-amino acid, a DNA for encoding N-carbamyl-D-amino acid hydrolase(“D-carbamylase”), a recombinant DNA containing the gene, a celltransformed with the recombinant DNA, a methods for producing a proteinusing the transformed cell and producing D-amino acid.

BACKGROUND OF THE INVENTION

In a known method for producing amino acid using an enzyme, a5-substituted hydantoin compound synthesized chemically at a low cost isemployed as a starting material and is decomposed asymmetrically intooptically active amino acids. A method for producing optically activeamino acid from such a 5-substituted hydantoin compound is important forproducing pharmaceuticals, chemical industrial products, food additivesand the like.

In a method for producing optically active amino acid from such a5-substituted hydantoin compound, the following enzymes (1) and (2) arerequired:

(1) An enzyme which catalyzes an N-carbamylamino acid producing reactionvia an action on a 5-hydantoin compound to hydrolyze this compound:hydantoin hydrolase (hydantoinase).

(2) An enzyme which catalyzes an optically active amino acid producingreaction via an action on a resultant N-carbamylamino acid to hydrolyzethis compound, N-carbamylamino acid hydrolase (carbamylase).

For producing optically active amino acid from a 5-substituted hydantoincompound describe above, an enzyme which is optically selective for atleast one of (1) hydantoinase and (2) carbamylase may be employed, andknown methods employ a microbial enzyme system or a microbial enzymesystem combined with a chemical reaction system.

Among such known methods, a known method for producing D-amino acid froma 5-substituted hydantoin compound using a D-amino acid producingmicroorganism or a material containing an enzyme produced by themicroorganism employs a Pseudomonas microorganism (Japanese PatentApplication Publication No. 56-003034) or an Agrobacterium microorganism(Japanese Patent Application Laid-open No. 019696). Such a D-amino acidproducing microorganism frequently has a hydantoinase activity specificgenerally to a 5-substituted hydantoin in a D form, and when using aDL-5-substituted hydantoin (5-benzylhydatoin as an example here) as astarting material, the D form is hydrolyzed exclusively to form anN-carbamyl-D-amino acid, which is then hydrolyzed by a D-carbamylasewhich acts exclusively on the D form, resulting in only an amino acid inthe D form (D-phenylalanine as an example here), as shown in thefollowing scheme.

SUMMARY OF THE INVENTION

Thus, a production of an optically active amino acid from a5-substituted hydantoin compound using a cultured cell of a D-amino acidproducing microorganism involves here a problem resulting from the needof a derivative such as a hydantoin derivative for the purpose ofincreasing the production of an enzyme required for the reaction, or theneed of a massive amount of the cultured cell.

On the other hand, for the purpose of an efficient production of anoptically active amino acid, it is preferable that a D-hydantoinase geneand a D-carbamylase gene are isolated and imparted with an increasedgene amplification, transcription and translation to obtain arecombinant whose ability of producing such an enzyme is enhanced, whichis then employed in the production. However, a conventional method isproblematically time consuming in effecting a reaction, and the reactionallows an N-carbamyl-D-amino acid to be formed as a by-product.

It is an object of the present invention to isolate a D-hydantoinasegene and a D-carbamylase gene from a microorganism having an ability ofconverting a 5-substituted hydantoin compound to a D-amino acid wherebyelucidating the amino acid sequence and the base sequence of an encodinggene and to construct a recombinant whose production of the enzyme isenhanced whereby providing a method for producing the D-amino acidefficiently from the 5-substituted hydantoin.

The present inventors made an effort for solving the problems describedabove, and were finally successful in isolating a D-hydantoinase geneand a D-carbamylase gene from a microorganism having an ability ofconverting a 5-substituted hydantoin compound into a D-amino acid,whereby establishing the present invention.

The DNA according to one aspect of the present invention has a basesequence represented by (a) or (b) and that encodes a protein havingD-hydantoinase activity, wherein

-   (a) is the base sequence represented by Seq. ID No. 1 in the    Sequence Listing; and-   (b) is a base sequence hybridizing with a complementary base    sequence of the base sequence represented by Seq. ID No. 1 in the    Sequence Listing under a stringent condition for encoding.

The DNA according to another aspect of the present invention has anamino acid sequence represented by (c) or (d) and that encodes a proteinhaving D-hydantoinase activity, wherein

-   (c) is the amino acid sequence represented by Seq. ID No. 2 in the    Sequence Listing;-   (d) is an amino acid sequence resulting from the substitution,    deletion, insertion, addition or inversion of one or several amino    acid residues in the amino acid sequence represented by Seq. ID No.    2 in the Sequence Listing.

The recombinant DNA according to a third aspect of the present inventionresults from a connection of a DNA according to the first aspect with avector DNA.

The recombinant DNA according to a fourth aspect of the presentinvention results from a connection of a DNA according to the secondaspect with a vector DNA.

The cell according to a fifth aspect is transformed with a recombinantDNA according to the third aspect.

The cell according to a sixth aspect is transformed with a recombinantDNA according to the fourth aspect.

The method for producing a protein having D-hydantoinase activityaccording to a seventh aspect comprises incubating a cell in a culturemedium, and allowing a protein having D-hydantoinase activity to beaccumulated in one or both of the culture medium and the cell. The cellbeing the cell according to the fifth aspect.

The method for producing a protein having D-hydantoinase activityaccording to an eighth aspect comprises incubating a cell in a culturemedium, and allowing a protein having D-hydantoinase activity to beaccumulated in one or both of the culture medium and the cell. The cellbeing the cell according to the sixth aspect.

The protein according to a ninth aspect of the present invention has anamino acid sequence represented by (a) or (b) and having D-hydantoinaseactivity, wherein

-   (a) is the amino acid sequence represented by Seq. ID No. 2 in the    Sequence Listing; and-   (b) is an amino acid sequence resulting from the substitution,    deletion, insertion, addition or inversion of one or several amino    acid residues in the amino acid sequence represented by Seq. ID No.    2 in the Sequence Listing.

The method for producing N-carbamyl-D-amino acid according to a tenthaspect of the present invention comprises producing a protein havingD-hydantoinase activity by the method according to the seventh aspect,and producing N-carbamyl-D-amino acid by making the protein havingD-hydantoinase activity react with a 5-substituted hydantoin. The cellbeing the cell according to the fifth aspect.

The method for producing N-carbamyl-D-amino acid according to aneleventh aspect of the present invention comprises producing a proteinhaving D-hydantoinase activity by the method according to the eighthaspect, and producing N-carbamyl-D-amino acid by making the proteinhaving D-hydantoinase activity react with a 5-substituted hydantoin. Thecell being the cell according to the sixth aspect.

The method for producing D-amino acid according to a twelfth aspect ofthe present invention comprises producing a protein havingD-hydantoinase activity by the method according to the seventh aspect,and producing D amino acid by making the protein having D-hydantoinaseactivity and an enzyme hydrolyzing an N-carbamyl-D-amino acid or amaterial containing the enzyme react with a 5-substituted hydantoin. Thecell being the cell according to the fifth aspect.

The method for producing D-amino acid according to a thirteenth aspectof the present invention comprises producing a protein havingD-hydantoinase activity by the method according to the eighth aspect,and producing D-amino acid by making the protein having D-hydantoinaseactivity and an enzyme hydrolyzing an N-carbamyl-D-amino acid or amaterial containing the enzyme react with a 5-substituted hydantoin. Thecell being the cell according to the sixth aspect.

The DNA according to a fourteenth aspect of the present invention has abase sequence represented by (a) or (b) and that encodes a proteinhaving D-carbamylase activity, wherein

-   (a) is the base sequence represented by Seq. ID No. 3 in the    Sequence Listing; and-   (b) is a base sequence hybridizing with a complementary base    sequence of the base sequence represented by Seq. ID No. 3 in the    Sequence Listing under a stringent condition.

The DNA according to a fifteenth aspect of the present invention has anamino acid sequence represented by (c) or (d) and that encodes a proteinhaving D-carbamylase activity, wherein

-   (c) is the amino acid sequence represented by Seq. ID No. 4 in the    Sequence Listing; and-   (b) is an amino acid sequence resulting from the substitution,    deletion, insertion, addition or inversion of one or several amino    acid residues in the amino acid sequence represented by Seq. ID No.    4 in the Sequence Listing.

The recombinant DNA according to a sixteenth aspect of the presentinvention results from a connection of the DNA according to thefourteenth aspect with a vector DNA.

The recombinant DNA according to a seventeenth aspect of the presentinvention results from a connection of the DNA according to thefifteenth aspect with a vector DNA.

The cell according to an eighteenth aspect of the present invention istransformed with a recombinant DNA according to the sixteenth aspect.

The cell according to a nineteenth aspect of the present invention istransformed with a recombinant DNA according to the seventeenth aspect.

The method for producing a protein having D-carbamylase activityaccording to a twentieth aspect of the present invention comprisesincubating a cell in a culture medium, and allowing a protein havingD-carbamylase activity to be accumulated in one or both of the culturemedium and the cell. The cell being the cell according to the eighteenthaspect.

The method for producing a protein having D-carbamylase activityaccording to a twenty-first aspect of the present invention comprisesincubating a cell in a culture medium, and allowing a protein havingD-carbamylase activity to be accumulated in one or both of the culturemedium and the cell. The cell being the cell according to the nineteenthaspect.

The protein according to a twenty-second aspect of the present inventionhas an amino acid sequence represented by (a) or (b) and havingD-carbamylase activity, wherein

-   (a) is the amino acid sequence represented by Seq. ID No. 4 in the    Sequence Listing; and-   (b) is an amino acid sequence resulting from the substitution,    deletion, insertion, addition or inversion of one or several amino    acid residues in the amino acid sequence represented by Seq. ID No.    4 in the Sequence Listing.

The method for producing D-amino acid according to a twenty-third aspectof the present invention comprises producing a protein havingD-carbamylase activity by the method according to the twentieth aspect,and producing D-amino acid by making the protein having D-carbamylaseactivity react with an N-carbamylamino acid. The cell being the cellaccording to the eighteenth aspect.

The method for producing D-amino acid according to a twenty-fourthaspect of the present invention comprises producing a protein havingD-carbamylase activity by the method according to the twenty-firstaspect, and producing D-amino acid by making the protein havingD-carbamylase activity react with an N-carbamylamino acid. The cellbeing the cell according to the nineteenth aspect.

The method for producing D-amino acid according to a twenty-fifth aspectof the present invention comprises producing a protein havingD-carbamylase activity by the method according to the twentieth aspect,and producing D-amino acid by making the protein having D-carbamylaseactivity and an enzyme hydrolyzing a 5-substituted hydantoin or amaterial containing the enzyme react with a 5-substituted hydantoin. Thecell being the cell according to the eighteenth aspect.

The method for producing D-amino acid according to a twenty-sixth aspectof the present invention comprises producing a protein havingD-carbamylase activity by the method according to the twenty-firstaspect, and producing D-amino acid by making the protein havingD-carbamylase activity and an enzyme hydrolyzing a 5-substitutedhydantoin or a material containing the enzyme react with a 5-substitutedhydantoin. The cell being the cell according to the nineteenth aspect.

The method for producing D-amino acid according to a twenty-seventhaspect of the present invention comprises producing a protein havingD-hydantoinase activity by the method according to the seventh aspect,the cell being the cell according to the fifth aspect, producing aprotein having D-carbamylase activity by the method according to thetwentieth aspect, the cell being the cell according to the eighteenthaspect, and producing D-amino acid by making the protein havingD-hydantoinase activity and the protein having D-carbamylase activityreact with a 5-substituted hydantoin.

The method for producing D-amino acid according to a twenty-eighthaspect of the present invention comprises producing a protein havingD-hydantoinase activity by the method according to the seventh aspect,the cell being the cell according to the fifth aspect, producing aprotein having D-carbamylase activity by the method according to thetwenty-first aspect, the cell being the cell according to the nineteenthaspect, and producing D-amino acid by making the protein havingD-hydantoinase activity and the protein having D-carbamylase activityreact with a 5-substituted hydantoin.

The method for producing D-amino acid according to a twenty-ninth aspectof the present invention comprises producing a protein havingD-hydantoinase activity by the method according to the eighth aspect,the cell being the cell according to the sixth aspect, producing aprotein having D-carbamylase activity by the method according to thetwentieth aspect, the cell being the cell according to the eighteenthaspect, and producing D-amino acid by making the protein havingD-hydantoinase activity and the protein having D-carbamylase activityreact with a 5-substituted hydantoin.

The method for producing D-amino acid according to a thirtieth aspect ofthe present invention comprises producing a protein havingD-hydantoinase activity by the method according to the eighth aspect,the cell being the cell according to the sixth aspect, producing aprotein having D-carbamylase activity by the method according to thetwenty-first aspect, the cell being the cell according to the nineteenthaspect, and producing D-amino acid by making the protein havingD-hydantoinase activity and the protein having D-carbamylase activityreact with a 5-substituted hydantoin.

Other objects and features of this invention will become apparent fromthe following description with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of the group of the genes encoding theD-hydantoinase and D-carbamylase of strains AJ11221, and

FIG. 2 is a flow chart of a process for producing D-hydantoinase andD-carbamylase according to the invention.

DETAILED DESCRIPTIONS

Embodiments of the present invention are described below in the order:

-   [I] DNA for encoding protein having D-hydantoinase activity and    protein having D-carbamylase activity,-   [II] Method for producing protein having D-hydantoinase activity and    protein having D-carbamylase activity, and-   [III] Method for producing D-amino acid.

In the following descriptions, a protein having D-hydantoinase activityis sometimes simply referred to as a D-hydantoinase. Moreover, a proteinhaving D-carbamylase activity is sometimes simply referred to as aD-carbamylase.

-   [I] DNA for encoding D-hydantoinase and D-carbamylase:

DNA for encoding D-hydantoinase and D-carbamylase according to theinvention is obtained by isolating it from a chromosomal DNA ofPasteurella pneumotropica strain AJ11221 (FERM-P4348) described inJapanese Patent Application Publication No. 56-015878. Pasteurellapneumotropica strain AJ11221 (FERM-P4348) was a microorganism which hadinitially been deposited under the name of Moraxella nonliquefaciens inNational Institute of Bioscience and Human-Technology, Ministry ofEconomy, Trade and Industry, METI, but was subsequently re-identified tobe classified in Pasteurella pneumotropica. Currently, Pasteurellapneumotropica has been deposited as FERM P-4348 with the ResearchInstitute of Bioscience and Human-technology, AIST, MITY, JAPAN, on Dec.20, 1977. Later, on May 30, 2002, the same strain has been deposited asFERM BP-8064 based on Budapest convention in the International PatentOrganism Depository (IPOD), National Institute of Advanced IndustrialScience and Technology (AIST), Japan (Tsukuba City, Higashi 1-1-1,Ibaraki Prefecture, Japan).

The microorganism described above was subjected to physiological andphysical tests in accordance with Bergey's Manual of DeterminativeBacteriology, Vol.1 (9th edition, 1994, Williams & Wilkins) which is atext for characterizing microorganisms, and the following results wereobtained.

Results of re-identification of Pasteurella pneumotropica strain AJ11221

Gram staining Negative Cell morphology Spherical baccilus Mobility NoneNitrate reduction + Indole production − Glucose acidificaiton − Argininedihydraze − Urease + Esculin hydrolysis − Gelatin hydrolysis −β-Galactosidase + Catalase + Oxidase + Substrate catabolyzing abilityGlucose − L-Arabinose − D-Mannose − D-Mannitol − N-Acetyl-D-glucosamine− Maltose + Potassium gluconate − n-Capric acid − Adipic acid − dl-Malicacid − Sodium citrate − Phenyl acetate −

Based on the bacteriological characteristics described above, AJ11221microorganism was identified as Pasteurella pneumotropica.

The present inventors were successful in obtaining a D-hydantoinase geneand D-carbamylase gene as being isolated from a gene library preparedusing chromosomal DNAs of the AJ11221 microorganism described above. Itis presumed that these genes are located at separated locations on thechromosome.

The method of a PCR employed for isolating the genes are described forexample in White, T. J. et al., Trends Genet. Vol.5, p. 185, 1989. Themethods for preparing a chromosomal DNA and for isolating an intendedDNA molecule from a gene library using a DNA molecule as a probe aredescribed for example in Molecular Cloning, 2nd edition, Cold SpringHarbor press (1989).

A method for sequencing a DNA for encoding the isolated D-hydantoinaseor D-carbamylase is described for example in A Practical Guide toMolecular Cloning, John Wiley & Sons, Inc. (1985). It is also possibleto determine a base sequence using a DNA sequencer manufactured byApplied Biosystems.

In the Sequence Listing attached hereto, the base sequence of the DNAfor encoding the D-hydantoinase derived from the AJ11199 microorganismcharacterized by the method described above is represented by Seq. IDNo. 1, while the DNA for encoding the D-carbamylase is represented bySeq. ID No. 3.

Each of these DNAs encodes a protein involved in the production of aD-amino acid.

In the Sequence Listing, Sequence ID No. 2 represents the amino acidsequence of the protein having D-hydantoinase activity encoded by thebase sequence of Sequence ID No. 1, while Sequence ID No. 4 representsthe amino acid sequence of the protein having D-carbamylase activityencoded by the base sequence of Sequence ID No. 3.

The protein having the D-hydantoinase activity represented by SequenceID No. 2 in the Sequence Listing and the protein having theD-carbamylase activity represented by Sequence ID No. 4 in the SequenceListing catalyze the reactions for forming an optically active aminoacid such as D-phenylalanine from a 5-substituted hydantoin such as5-benzylhydantoin, as shown in the following scheme.

A DNA for encoding a D-hydantoinase according to the invention and a DNAfor encoding a D-carbamylase are detailed below.

(1) DNA for Encoding D-hydantoinase

A D-hydantoinase gene according to the invention which has the basesequence represented by Sequence ID No. 1 in the Sequence Listing is oneisolated from a chromosomal DNA of Pasteurella pneumotropica strainAJ11221 as described above, and has the homology of 77% (76% in aminoacid sequence) to a known Agrobacterium microorganism-derivedD-hydantoinase gene (WO96/20275).

The DNA represented by Sequence ID No. 1 in the Sequence Listing DNA isnot the only DNA which encodes a D-hydantoinase according to theinvention. Thus, the base sequence differs among the species and thestrains of Pasteurella microorganisms.

The DNA according to the invention is not only a DNA for encoding theisolated D-hydantoinase but also a DNA resulting from an artificialvariation of the DNA for encoding the isolated D-hydantoinase, as amatter of course, as long as a D-hydantoinase is encoded. An artificialvariation method employed frequently is a site-specific variationintroducing method described for example in Method in Enzymol. P154,1987.

The DNA for encoding a protein having D-hydantoinase activity which hasa base sequence capable of hybridizing under a stringent condition witha complementary base sequence of the base sequence represented bySequence ID No. 1 in the Sequence Listing is also a DNA according to theinvention. The term “stringent condition” employed here means acondition allowing a specific hybrid to be formed but not allowing anon-specific hybrid to be formed. While it is difficult to specify sucha condition as definitive numerical parameters, those which may beexemplified are a condition which allows the DNAs which are highlyhomologous to each other, preferably have a homology of 80% or higher,more preferably a homology of 90% or higher to each other while notallowing any DNAs having a less homology and a condition of an ordinarysouthern hybridization washing step, i.e., a hybridization at a saltconcentration corresponding to 60° C., 1×SSC, 0.1% SDS, preferably 60°C., 0.1×SSC, 0.1% SDS. The term “D-hydantoinase activity” means anyactivity capable of producing N-carbamyl-D-amino acid by hydrolyzing a5-substituted hydantoin compound.

A DNA for encoding a protein which is identical substantially to theD-hydantoinase encoded by the DNA represented by Sequence ID No. 1 inthe Sequence Listing is also a DNA according to the invention. Thus, thefollowing (a) or (b) are in the scope of the present invention, where:

-   (a) is a DNA for encoding the amino acid sequence represented by    Seq. ID No. 2 in the Sequence Listing, and-   (b) is a DNA for encoding a protein having D-hydantoinase activity    which has an amino acid sequence resulting from the substitution,    deletion, insertion, addition or inversion of one or several amino    acid residues in the amino acid sequence represented by Seq. ID No.    2 in the Sequence Listing.

For the purpose of deducing, on the basis of the amino acid sequences(a) and (b) described above, a DNA for encoding such a sequence, a DNAbase sequence universal codon can be employed. The term “several” meansa number which results in no substantial deterioration of the stericstructure or enzymatic activity of the protein, and which is typically 2to 50, preferably 2 to 30, more preferably 2 to 10. The term“D-hydantoinase activity” means any activity capable of producingN-carbamyl-D-amino acid by hydrolyzing a 5-substituted hydantoincompound. Nevertheless, in the case of a protein involving such asubstitution, deletion, insertion, addition or inversion in the aminoacid sequence represented by Seq. ID No. 2 in the Sequence Listing, itis preferred that an enzymatic activity which is at least a half of thatof the protein having the amino acid sequence represented by Seq. ID No.2 in the Sequence Listing is possessed.

(2) DNA for Encoding D-carbamylase

A DNA for encoding the D-carbamylase according to the invention isdescribed below. The D-carbamylase of the present invention having thebase sequence represented by Sequence ID No. 3 in the Sequence Listingis isolated from a chromosomal DNA of Pasteurella pneumotropica strainAJ11221 and has the homology of 78% (81% in amino acid sequence) to aknown Agrobacterium microorganism-derived D-carbamylase gene (JapanesePatent Publication No. 2902112) and the homology of 67% (59% in aminoacid sequence) to a known Pseudomonas microorganism-derivedD-carbamylase gene (Japanese Patent Publication No. 2902112).

The DNA represented by Sequence ID No. 3 in the Sequence Listing DNA isnot the only DNA which encodes the D-carbamylase according to theinvention. Thus, the base sequence should differ among the species andthe strains of Pasteurella microorganisms.

A DNA resulting from an artificial variation of the DNA for encoding theisolated D-hydantoinase is also DNA of the present invention as long asit encodes a D-hydantoinase. An artificial variation method employedfrequently is a site-specific variation introducing method described forexample in Method in Enzymol. P154, 1987.

A DNA for encoding a protein having the D-carbamylase activity which hasa base sequence capable of hybridizing under a stringent condition witha complementary sequence of the base sequence represented by Sequence IDNo. 3 in the Sequence Listing is also a DNA according to the invention.The term “stringent condition” employed here means a condition allowinga specific hybrid to be formed but not allowing a non-specific hybrid tobe formed. While it is difficult to specify such a condition asdefinitive numerical parameters, those which may be exemplified are acondition which allows the DNAs which are highly homologous to eachother, preferably have a homology of 80% or higher, more preferably ahomology of 90% or higher to each other while not allowing any DNAshaving a less homology and a condition of an ordinary southernhybridization washing step, i.e., a hybridization at a saltconcentration corresponding to 60° C., 1×SSC, 0.1% SDS, preferably 60°C., 0.1×SSC, 0.1% SDS. The term “D-carbamylase activity” means anyactivity capable of producing D-amino acid by hydrolyzing anN-carbamyl-D-amino acid.

A DNA for encoding a protein which is identical substantially to theD-carbamylase encoded by the DNA described above is also a DNA accordingto the invention. Thus, the following (a) or (b) are in the scope of thepresent invention, where:

-   (a) is a DNA for encoding the amino acid sequence represented by    Seq. ID No. 4 in the Sequence Listing,-   (b) is a DNA for encoding a protein having D-hydantoinase activity    which has an amino acid sequence resulting from the substitution,    deletion, insertion, addition or inversion of one or several amino    acid residues in the amino acid sequence represented by Seq. ID No.    4 in the Sequence Listing.

For the purpose of deducing, on the basis of the amino acid sequences(a) and (b) described above, a DNA for encoding such a sequence, a DNAbase sequence universal codon can be employed. The term “several” meansa number which results in no substantial deterioration of the stericstructure or enzymatic activity of the protein, and which is typically 2to 50, preferably 2 to 30, more preferably 2 to 10. The term“D-carbamylase activity” means any activity capable of producing D-aminoacid by hydrolyzing an N-carbamyl-D-amino acid. Nevertheless, in thecase of a protein involving such a substitution, deletion, insertion,addition or inversion in the amino acid sequence represented by Seq. IDNo. 4 in the Sequence Listing, it is preferred that an enzymaticactivity which is at least a half of that of the protein having theamino acid sequence represented by Seq. ID No. 4 in the Sequence Listingis possessed.

[III] Method for Producing D-hydantoinase and D-carbamylase:

A method for producing D-hydantoinase and D-carbamylase by a recombinantDNA technology is discussed below. A large number of the methods forproducing useful proteins such as enzymes or physiologically activesubstances utilizing recombinant DNA technologies are known, and a useof the recombinant DNA technology allows a useful protein, which occursnaturally only in a trace amount, to be produced on a large scale.

FIG. 2 shows a flow chart of a process for producing D-hydantoinase andD-carbamylase according to the invention.

First, D-hydantoinase DNA of the present invention and/or D-carbamylaseDNA are prepared (Step S1).

Then, the prepared NDA is attached to a vector DNA to prepare arecombinant DNA (Step S2), and then a cell is transformed with therecombinant DNA to give a transformant (Step S3). Subsequently, thetransformant is incubated in a culture medium to allow theD-hydantoinase and/or D-carbamylase to be produced and accumulated inthe culture medium (Step S4).

In the following Step S5, the enzyme is recovered and purified, wherebyaccomplishing a large scale production of the D-hydantoinase and/orD-carbamylase.

An amino acid synthesis using the enzyme produced in Step S5 and theculture medium of Step S4 in which the enzyme in accumulated enables alarge scale production of an intended amino acid (Step S6).

A DNA to be attached to a vector DNA may be any one allowing aD-hydantoinase of the present invention and/or D-carbamylase to beexpressed.

A D-hydantoinase gene employed here to be attached to a vector DNA mayfor example be those already described above, that is:

-   (a) is the DNA having the base sequence represented by Seq. ID No. 1    in the Sequence Listing,-   (b) is the DNA having a base sequence hybridizing with a    complementary base sequence of the base sequence represented by Seq.    ID No. 1 in the Sequence Listing under a stringent condition,-   (c) is the DNA for encoding the amino acid sequence represented by    Seq. ID No. 2 in the Sequence Listing, and-   (d) is the DNA for encoding an amino acid sequence resulting from    the substitution, deletion, insertion, addition or inversion of one    or several amino acid residues in the amino acid sequence    represented by Seq. ID No. 2 in the Sequence Listing.

A D-carbamylase gene employed here to be attached to a vector DNA mayfor example be those already described above, that is:

-   (a) is the DNA having the base sequence represented by Seq. ID No. 3    in the Sequence Listing,-   (b) is the DNA having a base sequence hybridizing with a    complementary base sequence of the base sequence represented by Seq.    ID No. 3 in the Sequence Listing under a stringent condition,-   (c) is the DNA for encoding the amino acid sequence represented by    Seq. ID No. 4 in the Sequence Listing, and-   (d) is the DNA for encoding an amino acid sequence resulting from    the substitution, deletion, insertion, addition or inversion of one    or several amino acid residues in the amino acid sequence    represented by Seq. ID No. 4 in the Sequence Listing.

In addition to the DNAs listed above, a DNA in which a D-hydantoinasegene is ligated to a D-carbamylase may also be employed. In such a case,D-hydantoinase of the present invention and the D-carbamylase of thepresent invention are expressed simultaneously.

When a recombinant DNA technology is employed to produce a protein on alarge scale, a host cell to be transformed may for example be abacterial cell, actinomyces cell, yeast cell, fungal cell, plant cell,animal cell and the like. Since technologies for producing proteins onlarge scales using coliform microorganisms have extensively beenreported, such a coliform microorganism, especially Escherichia coli, isemployed generally. A method for producing D-hydantoinase and/orD-carbamylase using a transformed coliform microorganism is discussedbelow.

A promoter for expressing a DNA for encoding a D-hydantoinase and/orD-carbamylase may be a promoter employed usually for producing a proteinin a coliform microorganism, which may for example be a potent promotersuch as a T7 promoter, trp promoter, lac promoter, tac promoter, PLpromoter and the like.

Also for the purpose of increasing the production, it is preferable toligate a terminator which is a transcription termination sequence to thedownstream of the protein gene. Such a terminator may for example be aT7 terminator, fd phage terminator, T4 terminator, tetracyclin resistantgene terminator, coliform trpA gene terminator and the like.

A vector for transducing a gene encoding a hydantoinase and/orD-carbamylase into a host cell is preferably be one of a multicopy type,such as a plasmid having a replication initiating point derived from ColE1,for example, a pUC plasmid or pBR322 plasmid or a derivative thereof.The term “derivative” employed here is a plasmid which has beensubjected to an alteration by the substitution, deletion, insertion,addition or inversion of bases. The alteration referred here means toinclude an alteration by a mutating treatment using a mutating agent orUV irradiation as well as a spontaneous mutation.

For the purpose of screening for a transformant, the vector having amarker such as an ampicillin resistant gene is employed preferably, suchas a plasmid available as an expression vector having a potent promoter,for example, a pUC (Takara Shuzo Co., Ltd.), pPROK (Clonetech), pKK233-2(Clonetech) and the like.

A DNA fragment in which a promoter, a gene encoding a hydantoinaseand/or D-carbamylase and a terminator are ligated in this order is thenligated to a vector DNA to obtain a recombinant DNA.

The recombinant DNA is employed to transform a host cell, and then thecell is incubated, resulting in the expression and production of thehydantoinase and/or D-carbamylase. As a method for effecting atransformation and a method for screening for a transformant, thosedescribed in Molecular Cloning, 2nd edition, Cold Spring Harbor press(1989) are applicable.

As a production medium, a culture medium employed ordinarily forincubating a coliform microorganism such as a M9-casamino acid mediumand LB medium may be employed. The incubation condition and theproduction inducing condition are selected appropriately depending onthe types of the marker of the vector, promoter and host cell employed.In order to increase the production of the enzyme, it is also preferredto add isopropyl 1-thio-β-D-galactopyranoside (IPTG) to the culturemedium or to conduct an enzyme inducing treatment such as warming.

After recovering a cultured cell for example by a centrifugation, thecell is crushed or subjected to lysis to recover the hydantoinase and/orD-carbamylase, which can be used as a crude enzyme solution. The cellcan be crushed by an ultrasonication, French press crushing, glass beadcrushing, and the like, or may be subjected to lysis using an albumenlysozyme, peptitase treatment, or a combination thereof. If necessary,the enzyme may be purified by an ordinary procedure such as aprecipitation, filtration and column chromatography. In such a case, apurification utilizing an antibody against the enzyme itself can also beutilized.

A D-hydantoinase according to the invention obtained by a recombinantdescribed above is a protein having D-hydantoinase activity which has anamino acid sequence represented by (a) or (b), where:

-   (a) is the amino acid sequence represented by Seq. ID No. 2 in the    Sequence Listing,-   (b) is the amino acid sequence resulting from the substitution,    deletion, insertion, addition or inversion of one or several amino    acid residues in the amino acid sequence represented by Seq. ID No.    2 in the Sequence Listing.

A D-carbamylase according to the invention obtained by a recombinantdescribed above is a protein having D-carbamylase activity which has anamino acid sequence represented by (c) or (d), where:

-   (c) is the amino acid sequence represented by Seq. ID No. 4 in the    Sequence Listing,-   (d) is the amino acid sequence resulting from the substitution,    deletion, insertion, addition or inversion of one or several amino    acid residues in the amino acid sequence represented by Seq. ID No.    4 in the Sequence Listing.

The definitions of the terms “several”, “D-hydantoinase activity” and“D-carbamylase activity” employed here are synonymous with thosedescribed in [I] DNA for encoding D-hydantoinase and D-carbamylase.

[III] Method for Producing D-amino Acid

A method for producing D-amino acid employing a hydantoinase and/orD-carbamylase according to the invention is discussed below.

A method for producing D-amino acid according to the invention employsthe enzymes of the present invention as at least one of hydantoinase andcarbamylase, the combination of which may be one of the three shownbelow. That is,

-   (i) D-hydantoinase of the present invention+carbamylase,-   (ii) Hydantoinase +carbamylase of the present invention, and-   (iii) D-hydantoinase of the present invention+carbamylase of the    present invention.

In the case of combination (i), a D-hydantoinase may for example be aprotein having D-hydantoinase activity which has an amino acid sequencerepresented by (a) or (b), where:

-   (a) is the amino acid sequence represented by Seq. ID No. 2 in the    Sequence Listing,-   (b) is the amino acid sequence resulting from the substitution,    deletion, insertion, addition or inversion of one or several amino    acid residues in the amino acid sequence represented by Seq. ID No.    2 in the Sequence Listing.

It is also possible that a DNA for encoding such a D-hydantoinase isattached to a vector to form a recombinant DNA by which a cell is thentransformed and the cell is incubated to yield a D-hydantoinase for use.When a transformed cell is used to produce a D-hydantoinase, a substratemay be added directly to the culture medium during the incubation, orthe cell or washed cell recovered from the culture medium may beemployed. A cell treatment product obtained by crushing or beingsubjected to lysis may be employed as it is, or a D-hydantoinase may berecovered from the cell treatment product and used as a crude enzymesolution or maybe purified before use. Thus, any fraction containingD-hydantoinase activity can be employed.

A substrate for a D-hydantoinase according to the invention may be any5-substituted hydantoin compound capable of being hydrolyzed at thesubstrate specificity of this enzyme. Those which may be exemplified area 5-substituted hydantoin compound corresponding to a natural amino acidsuch as hydantoin, 5-methylhydantoin, 5-benzylhydantoin,5-(4-hydroxybenzyl)hydantoin, 5-indolylmethylhydantoin,5-(3,4-dihydroxybenzyl)hydantoin, 5-methylthioethylhydantoin,5-isopropylhydantoin, 5-isobutylhydantoin, 5-sec-butylhydantoin,5-(4-aminobutyl)hydantoin, 5-hydroxymethylhydantoin and the like, aswell as a 5-substituted hydantoin compound corresponding to anon-natural amino acid or a derivative thereof, such as5-phenylhydantoin, 5-(4-hydroxyphenyl)hydantoin,5-methoxymethylhydantoin, 5-benzyloxymethylhydantoin,5-(3,4-methylenedioxybenzyl) hydantoin, dihydrouracil and the like.

A carbamylase to be combined with a D-hydantoinase of the presentinvention may be any known enzyme capable of catalyzing a reaction whicheffects hydrolitycally on an N-carbamyl-D-amino acid to yield a D-aminoacid or a material containing such an enzyme. Thus, it may be acarbamylase acting specifically on an N-carbamyl-D-amino acid(D-carbamylase) or a non-optically selective carbamylase. The term“material containing an enzyme” employed here means a materialcontaining the enzyme, such as those containing a culture medium,cultured cell, cell treatment product obtained by crushing the cell orsubjecting the cell to a lysis, crude enzyme solution or purifiedenzyme.

A D-carbamylase is known to be present for example in Pseudomonas orAgrobacterium microorganisms (Japanese Patent 2902112).

Combination (ii) is discussed below. AD-carbamylase of the presentinvention may for example be a protein having D-carbamylase activitywhich has an amino acid sequence represented by (a) or (b), where:

-   (a) is the amino acid sequence represented by Seq. ID No. 4 in the    Sequence Listing,-   (b) is the amino acid sequence resulting from the substitution,    deletion, insertion, addition or inversion of one or several amino    acid residues in the amino acid sequence represented by Seq. ID No.    4 in the Sequence Listing.

It is also possible that a DNA for encoding such a D-carbamylase isattached to a vector to form a recombinant DNA by which a cell is thentransformed and the cell is incubated to yield a D-carbamylase for use.When a transformed cell is used to produce a D-carbamylase, a substratemay be added directly to the culture medium during the incubation, orthe cell or washed cell recovered from the culture medium may beemployed. A cell treatment product obtained by crushing or beingsubjected to lysis may be employed as it is, or a D-carbamylase may berecovered from the cell treatment product and used as a crude enzymesolution or may be purified before use. Thus, any fraction containingD-carbamylase activity can be employed.

A substrate for a D-carbamylase according to the invention may be anyN-carbamyl-D-amino acid capable of being hydrolyzed at the substratespecificity of this enzyme. Thus, an N-carbamyl-D-amino acid other thanthe N-carbamyl-D-amino acids obtained from the 5-substituted hydantoincompounds listed above can also be employed as a substrate.

A hydantoinase to be combined with a D-carbamylase of the presentinvention may be any known enzyme capable of catalyzing a reaction whicheffects hydrolitycally on a 5-substituted hydantoin compound to yield anN-carbamylamino acid or a material containing such an enzyme. The term“material containing an enzyme” employed here means a materialcontaining the enzyme, such as those containing a culture medium,cultured cell, cell treatment product obtained by crushing the cell orsubjecting the cell to a lysis, crude enzyme solution or purifiedenzyme. Nevertheless, since a D-carbamylase of the present invention isD-form-specific, a non-optically specific hydantoinase or aD-hydantoinase acting specifically on a D form should be employed. Anon-optically specific hydantoinase known to be present for example inMicrobacterium liquefaciens strain AJ3912 (Japanese Patent ApplicationNo. 2001-298619) A D-hydantoinase which acts specifically on a hydantoinin the D form is known to be present for example in Agrobacterium sp.Strain AJ11220 (Japanese Patent Application Publication No. 56-003034).Microbacterium liquefaciens strain AJ3912 was a microorganism which wasdeposited on Jun. 27, 1975 in National Institute of Bioscience andHuman-Technology, Ministry of Economy, Trade and Industry, METI, andreceived the acceptation number FERM-P3133. Later, on Jun. 27, 2001, thesame strain has been deposited as FERM BP-7643 based on Budapestconvention. Agrobacterium sp. Strain AJ11220 (FERM-P4347) was amicroorganism which had initially been deposited under the name ofPseudomonas sp. on Dec. 20, 1977 in National Institute of Bioscience andHuman-Technology, Ministry of Economy, Trade and Industry, METI, but wassubsequently re-identified to be classified in Agrobacterium sp.Currently, it is deposited in the International Patent OrganismDepositary of National Institute of Advanced Industrial Science andTechnology as Agrobacterium sp. Strain AJ11220 (National deposit No.FERM-P4347, International deposit No. FERM BP-7645).

In Combination (iii), a D-hydantoinase of the present inventiondescribed in the section of Combination (i) and a D-carbamylase of thepresent invention described in the section of Combination (ii) arecombined. Among Combination (I) to (iii), the most preferred combinationis Combination (iii).

In the reaction process employed here, a mixture of a D-hydantoinase andD-carbamylase may act on a 5-substituted hydantoin compound, or aD-hydantoinase may first act on a 5-substituted hydantoin compound whichis then subjected to the action of a D-carbamylase. The former processis preferred for the purpose of the simplification of the reactionprocess.

When producing D-amino acid by a method employing any of Combination (i)to (iii) described above, it is possible to produce an N-carbamylaminoacid or amino acid in the L form. For example, a D-hydantoinase of thepresent invention is employed to produce an N-carbamyl-D-amino acid froma DL-5-substituted hydantoin, and then the remaining L-5-substitutedhydantoin is separated from the N-carbamyl-D-amino acid to recover theL-5-substituted hydantoin, which is then hydrolyzed to yield anN-carbamyl-L-amino acid, which is further hydrolyzed to yield an L-aminoacid. Although such a hydrolyzing reaction may employ a hydrolyzingenzyme acting on an L form, a chemical hydrolyzing treatment for examplewith nitrous acid also enables the production of an L-amino acid at ahigh yield with preserving the optical activity.

When converting a DL-5-substituted hydantoin compound into a D-aminoacid, the D-amino acid can be produced at a molar yield of 50% or higherfrom the DL-5-substituted hydantoin compound by means of a combinationof the spontaneous racemization or chemical racemization of a5-substituted hydantoin compound, or the racemization employing ahydantoin racemase.

In other words, it is preferred to use a hydantoin racemase in additionto a D-hydantoinase and D-carbamylase. Such a hydantoin racemase ispreferably the hydantoin racemase derived from Microbacteriumliquefaciens strain AJ3912 (FERM-P3133) described in Japanese PatentApplication 2001-278739. In such a case, a D-amino acid can be producedtheoretically at 100% molar yield from a DL-5-substituted hydantoincompound since a hydantoin racemase contained in a protein mixturecatalyzes the racemization of the 5-substituted hydantoin compound asshown in the following scheme.

It is also possible to produce an N-carbamylamino acid using the proteinmixture described above. For example, the N-carbamylamino acid can beproduced by interrupting the hydrolysis reaction at the stage of theN-carbamylamino acid by adding, for example, a D-carbamylase inhibitorto the protein mixture described above.

When an amino acid producing reaction is allowed to proceed using aculture medium of a cell transformed by a recombinant DNA obtained byligating a DNA for encoding a D-hydantoinase of the present inventionand/or D-carbamylase with a vector, isolated cell, washed cell, celltreatment product, crude enzyme solution or purified enzyme solutionobtained from the cell treatment product, then a reaction mixturecontaining the 5-substituted hydantoin compound and the culture medium,isolated cell, washed cell, cell treatment product, crude enzymesolution or purified enzyme solution is kept at an appropriatedtemperature of 25 to 60° C. at pH 5 to 9 while allowing to stand or stirfor 8 hours to 5 days.

When an amino acid producing reaction is allowed to proceed whileincubating a cell transformed by a recombinant DNA obtained by ligatinga DNA for encoding a D-hydantoinase of the present invention and/orD-carbamylase with a vector in an aqueous medium, an aqueous mediumcontaining a 5-substituted hydantoin compound together with nutrientcomponents essential for the growth of the transformed cell such ascarbon sources, nitrogen sources, inorganic ions and the like isemployed. The addition of organic trace nutrient components such asvitamins and amino acids may frequently lead to a satisfactory result.It is also possible to add the 5-substituted hydantoin compound inportions. It is preferred to conduct the incubation in an aerobiccondition at pH 5 to 9 at an appropriated temperature of 25 to 40° C.for 8 hours to 5 days.

The D-amino acid in a culture medium a reaction mixture can rapidly bequantified by a known method. Thus, for convenience, a thin layerchromatography using for example a HPTLC CHIR manufactured by Merck canbe utilized, and, for obtaining a further higher analytical accuracy, ahigh pressure liquid chromatography (HPLC) employing an opticalresolution column such as a CHIRALPAK WH manufactured by DAICEL CHEMICALINDUSTRIES, LTD. can be employed.

A D-amino acid accumulated in a culture medium or reaction mixture canbe collected from the culture medium or reaction mixture by a standardmethod. For example, procedures such as filtration, centrifugation,concentration under vacuum, ion exchange or adsorption chromatography,crystallization and the like, may be employed if necessary incombination with each other. Especially after converting from a5-substituted hydantoin compound at a high concentration, the D-aminoacid can readily be isolated as a crystal by cooling the culture mediumor reaction mixture and adjusting the pH.

The present invention is further described in the following examples,which are not intended to restrict the invention. The quantification andthe optical purity assay of a 5-substituted hydantoin compound,N-carbamylamino acid and amino acid in an example are conducted usingHPLC employing an optical resolution column, CHIRALPAK WH, manufacturedby DAICEL CHEMICAL INDUSTRIES, LTD. The analytical condition isdescribed below.Column, CHIRALPAK WH 0.46 cm φ×25 cm (DAICEL CHEMICAL INDUSTRIES, LTD.)

-   Mobile phase: 5% (v/v) Methanol, 1 mM CuSO₄-   Column temperature: 50° C.-   Flow rate: 1.5 ml/min-   Detection: UV₂₁₀

EXAMPLE 1 Isolation of D-hydantoinase Gene and D-carbamylase Gene fromAJ11221 Microorganism

1. Cell Preparation

Pasteurella pneumotropica strain AJ11221 was incubated in a CM2G agarmedium (glucose 0.5%, yeast extract 1.0%, peptone 1.0%, NaCl 0.5%, Agar2%, pH 7.0) at 30° C. for 24 hours, whereby being refreshed. One“spatulaful” of the culture medium was inoculated to a 500 mL Sakaguchiflask containing 50 ml of a CM2G liquid medium, which was then incubatedaerobically with shaking at 30° C. for 16 hours.

2. Collection of Chromosomal DNA from Cell

50 ml of the culture medium was centrifuged (12,000×G, 4° C., 15minutes) and the cell was collected. The cell was then washed bysuspending in 10 ml of 50:20 TE (50 mM Tris-HCl, (pH 8.0), 20 mM EDTA)and recovered by centrifugation, and then re-suspended in 10 ml of 50:20TE. To this suspension, 0.5 ml of a 20 mg/ml lysozyme solution and 1 mlof a 10% SDS solution were added, and the mixture was incubated at 55°C. for 20 minutes. After the incubation, one volume of 10:1 TE-saturatedphenol was added to remove proteins. To the separated water layer, onevolume of 2-propanol was added to precipitate a DNA for recovery. Theprecipitated DNA was dissolved in 0.1 ml of 50:20 TE, to which 5 μl of10 mg/ml RNase and 5 μl of 10 mg/ml Proteinase K were added, and themixture was reacted at 55° C. for 20 minutes. After the reaction,proteins were removed using one volume of 10:1 TE-saturated phenol. Tothe separated water layer, one volume of 24:1 chloroform/isoamyl alcoholwas added, and the mixture was stirred and then the water layer wasrecovered. This procedure was repeated twice and then the resultantwater layer was supplemented with a 3M sodium acetate solution (pH 5.2)at the final concentration of 0.4 M, and two volumes of ethanol was thenadded. The precipitated DNA was recovered, washed with 70% ethanol,dried, and then dissolved in 1 ml of 10:1 TE.

3. Isolation of D-carbamylase from Gene Library

200 μg of the chromosomal DNA of Pasteurella pneumotropica strainAJ11221 was supplemented with 1U of a restriction enzyme Sau3AI, and themixture was reacted at 37° C. for 15 minutes to effect a partialdigestion. Then, from this DNA 3 to 8 kbp fragments were recovered by anagarose electrophoresis. The fragments were ligated with BamHI digestionproducts of a plasmid pUC18, whereby transforming an Esherichia coilstrain JM109 to prepare a gene library. The library was plated onto anampicillin-supplemented LB medium (tryptone 1%, yeast extract 0.5%,sodium chloride 1%, ampicillin 0.01%, agar 2%, pH 7.0), and then acolony was inoculated in a liquid medium containing as the only nitrogensource N-carbamyl-D-phenylalanine (glucose 0.2%,N-carbamyl-D-phenylalanine 0.2%, Na₂HPO₄0.6%, KH₂PO₄0.3%, NaCl 0.05%,MgSO₄ 0.012%, CaCl₂ 0.1 mM, ampicillin 0.01%, thiamine 0.0001%, pH 7.0)to effect an enrichment culture and then a strain capable of growingwith N-carbamyl-D-phenylalanine as the only nitrogen source wasselected. A transformant thus obtained was isolated and subjected to anincubation on an LB medium containing ampicillin andisopropyl-1-thio-β-D-galactopyranoside (IPTG), followed bycentrifugation and cell collection, and then the cell was added at 1% toa 0.1 M phosphate buffer solution containing 0.5%N-carbamyl-D-phenylalanine and allowed to react at 37° C. for 5 hours.The reaction mixture was analyzed and D-phenylalanine was revealed to beproduced, whereby assuring that this transformant contained the plasmidcontaining the intended gene. From this transformant, a plasmid DNA wasprepared and designated as pUC413-1.

4. Base Sequence of Inserted Fragment

The base sequence of an inserted fragment of the plasmid pUC413-1 wasdetermined by a dideoxy method, and is shown as Sequence ID No. 5 in theSequence Listing. As a result, the length of the inserted fragment was2.8 kbp and the open reading frame of about 0.9 kb (ORF1; Base No. 1141to 2064) was proven to be contained (FIG. 1).

5. Homology Between ORF1 and Known Sequence

ORF1 was characterized for the homology to known sequences, and provento exhibit the homology of 78% (81% in amino acid sequence) to a knownAgrobacterium microorganism-derived D-carbamylase gene and the homologyof 67% (59% in amino acid sequence) to Pseudomonas microorganism-derivedD-carbamylase gene. As a result, ORF1 was assumed to contain aD-carbamylase gene. However, any sequence exhibiting a homology to aD-hydantoinase was not found. The base sequence of the entire length ofthe D-carbamylase is shown as Sequence ID No. 3 in the Sequence Listing,and the corresponding amino acid sequence is shown as Sequence ID No. 4in the Sequence Listing.

6. Isolation of D-hydantoinase Gene From Gene Library

Accordingly, a D-hydantoinase gene was isolated using a gene library ofPasteurella pneumotropica strain AJ11221 prepared in Section 3 describedabove. For a screening, a transformant was cultured on an LB platecontaining ampicillin, transferred to a pH indicator-supplementedscreening plate (D-5-benzylhydantoin 0.5%, cresol red 0.005%, MnCl₂ 1mM, agar 2%, pH 8.5), and screened for a strain exhibiting the change inthe color of the pH indicator in response to the production ofN-carbamyl-D-phenylalanine. This transformant was used to prepare aplasmid containing a D-hydantoinase gene, which was then designated aspUC413-2.

7. Base sequence of Inserted Fragment

The base sequence of an inserted fragment of the plasmid pUC413-2 wasdetermined by a dideoxy method, and is shown as Sequence ID No. 6 in theSequence Listing. As a result, the length of the inserted fragment was2.3 kbp and the open reading frame of about 1.4 kb (ORF2; Base No. 184to 1572) was proven to be contained (FIG. 1).

8. Homology Between ORF2 and Known Sequence

ORF2 was characterized for the homology to known sequences, and provento exhibit the homology of 77% (76% in amino acid sequence) to aAgrobacterium microorganism-derived D-hydantoinase gene. As a result,ORF2 was assumed to contain a D-hydantoinase gene. The base sequence ofthe entire length of the D-hydantoinase is shown as Sequence ID No. 1 inthe Sequence Listing, and the corresponding amino acid sequence is shownas Sequence ID No. 2 in the Sequence Listing.

EXAMPLE 2 Expression of Strain AJ11221-Derived D-hydantoinase Gene andD-carbamylase Gene in E.coli

1. Construction of Expression Plasmid

In order to express the both genes in E.coli, plasmids pUC413H andpUC413C in which the both genes were ligated to the downstream of thelac promoter of pUC18 were constructed as follows. First, a chromosomalDNA of Pasteurella pneumotropica strain AJ11221 was employed as atemplate to amplify each gene by a PCR using the oligonucleotides shownin Table 1 as primers. Each of these fragments was digested withXbaI/HindIII and EcoRI/XbaI and ligated to the XbaI/HindIII andEcoRI/XbaI digestion product of pUC18, and then transduced into E.coliJM109. The ampicillin resistant strains were screened for strains havingintended plasmids, which were designated as expression plasmids pUC413Hand pUC413C.

TABLE 1 Primers employed for amplifying D-hydantoinase gene andD-carbamylase gene derived from strain AJ11221 Hydantoinase 5′-endCGCTCTAGAGGGAGACTGACGATGGATCTCATCGT Sequence ID No. 7    XbaI Initiationcodon 3′-end CGCAAGCTTCCTCTGACGCGGCGAATG Sequence ID No. 8     HindIIICarbamylase 5′-end CGCGAATTCCATCGAACCAGGGAGGATTTTGGA Sequence ID No. 9    EcoRI 3′-end CGCTCTAGACGCCCGCTAGCGGACCTGTT Sequence ID No. 10   XbaI Termination codon2. Preparation of Cell-Free Extract

Each of an E.coli transformant having pUC632H and an E.coli transformanthaving pUC632C was seed-cultured for 16 hours at 37° C. in an LB mediumcontaining 0.1 mg/ml ampicillin. 1 ml of this seed culture was added toa 500 ml Sakaguchi flask containing 50 m of an LB medium, to which 1 mlof the seed culture was added, and the mixture was major-cultured at 37°C. 2.5 hours after initiating the incubation, IPTG was added at thefinal concentration of 1 mM, and the incubation was continued furtherfor 4 hours.

After completing the incubation, the cells were collected, washed, andsuspended in 5 ml of 50 mM KBP (pH 8,0) and crushed using bead heaterfor 3 minutes (30 seconds×6 cycle, at 90 seconds interval) together with0.1 mm φ glass beads. The solution was recovered, centrifuged at 20,000G for 10 minutes, and the supernatant was obtained as a cell-freeextract.

3. D-hydantoinase and D-carbamylase Activity Assay

The assay of the D-hydantoinase activity was conducted by incubating areaction mixture containing 120 mg/dl D-5-benzylhydantoin (BH), 50 mMKPB (pH 8.0) and an enzyme solution at 37° C. for 30 minutes, adding 9volumes of 1.1 mMCuSO₄, 11.1 mM H₃PO₄, centrifuging at 20,000G for 10minutes to remove the pellet, and then quantifying the resultantN-carbamylphenylalanine (N—Car—Phe) by HPLC. One unit of the enzymeactivity was defined as an enzymatic activity capable of producing 1μmol of N-carbamylphenylalanine per 1 minute under the conditiondescribed above.

The assay of the D-carbamylase activity was conducted by incubating areaction mixture containing 80 mg/dl N-carbamyl-D-phenylalanine, 50 mMKPB (pH 7.5) and an enzyme solution at 37° C. for 30 minutes, adding 9volumes of 1.1 mMCuSO₄, 11.1 mM H₃PO₄, centrifuging at 20,000G for 10minutes to remove the pellet, and then quantifying the resultantN-phenylalanine (D—Phe) by HPLC. One unit of the enzyme activity wasdefined as an enzymatic activity capable of producing 1 μmol ofN-phenylalanine per 1 minute under the condition described above.

The results are shown in Table 2. Since the strain transformed bypUC413H exhibited D-hydantoinase activity and the strain transformed bypUC413C exhibited D-carbamylase activity, it was proven that the bothgenes were a D-hydantoinase gene and D-carbamylase gene derived fromPasteurella pneumotropica strain AJ11221 and were expressed in the cellof E.coli.

TABLE 2 Enzymatic activity of cell-free extract of E. coli havingpUC413H and pUC413C D-Hydantoinase D-Carbamylase IPTG activity activityPlasmid supplement (U/mg) (U/mg) pUC413H + 0.11 Not detected − 0.01 Notdetected pUC413C + Not detected 0.15 − Not detected 0.02 pUC18 + Notdetected Not detected − Not detected Not detected

EXAMPLE 3 Production of D-phenylalanine Using Washed E.coli Cell

Washed cells of JM109/pUC413H and JM109/pUC413C incubated similarly toExample 2 were prepared, added each at 1 g/dl to 0.1 mM KPB (pH 7.5)containing 1 g/dl of D-5-benzylhydantoin and reacted at 30° C. Bysampling at 24 hours after the reaction followed by centrifugationfollowed by analysis of the supernatant by HPLC, D-phenylalanineproduced was quantified.

The results are shown in Table 3. As evident from this table, by usingthe washed E.coli cells in which the D-hydantoinase gene andD-carbamylase gene were expressed, D-phenylalanine was producedefficiently from benzylhydantoine.

TABLE 3 D-Phenylalanine production using washed E. coli cellsD-Hydantoinase D-Carbamylase Phenylalanine gene-expressinggene-expressing production level strain strain (g/dl) JM109/pUC413HJM109/pUC413C 0.31 JM109/pUC18 JM109/pUC18 0.00

EXAMPLE 4 Production of D-amino Acid Using Washed E.coli Cell

Washed cells of JM109/pUC413H and JM109/pUC413C prepared similarly toExample 3 were added each at 1 g/dl to 0.1 mM KPB (pH 7.5) containing 1g/dl of each of 5-substituted hydantoin compounds, and reacted at 30° C.By sampling 24 hours after the reaction followed by centrifugationfollowed by analysis of the supernatant by HPLC, the resultant eachamino acid was quantified.

The results are shown in Table 4. As evident from this table, by usingthe washed E.coli cells in which the D-hydantoinase gene andD-carbamylase gene were expressed, each D-amino acid was producedefficiently from each 5-substituted hydantoin compound.

TABLE 4 D-Amino acid production using washed E. coli cells 5-SubstitutedProduced Production hydantoin amino level compound acid (g/dl)D-5-(4-Hydroxybenzyl)hydantoin D-Tyrosine 0.12D-5-Indolylmethylhydantoin D-Tryptophan 0.10D-5-Methylthioethylhydantoin D-Methionine 0.28 Hydantoin D-Glycine 0.08D-5-Methylhydantoin D-Alanine 0.03 D-5-Isopropylhydantoin D-Valine 0.06D-5-Isobutylhydantoin D-Leucine 0.01 D-5-sec-Butylhydantoin D-Isoleucine0.01 D-5-(4-Aminobutyl) hydantoin D-Lysine 0.05D-5-Carboxyethylhydantoin D-Glutamic acid 0.04 D-5-PhenylhydantoinD-Phenylglycine 0.30 D-5-(4-Hydroxyphenyl)hydantoin D-4-Hydroxyphenyl0.23 glycine

EXAMPLE 5 D-phenylalanine Production when Combined with Racemizationwith Hydantoin Racemase

An E.coli transformant having a plasmid pUCFHR containing a hydantoinracemase gene derived from Microbacterium liquefaciens strain AJ3912(FERM-P3133) described in Japanese Patent Application No. 2001-278739was seed-cultured for 16 hours at 37° C. in an LB medium containing 0.1mg/ml of ampicillin. 1 ml of this seed culture was added to a 500 mlSakaguchi flask containing 50 ml of an LB medium, to which 1 ml of theseed culture was added, and the mixture was major-cultured at 37° C. 2.5hours after initiating the incubation, IPTG was added at the finalconcentration of 1 mM, and the incubation was continued further for 4hours. After completing the incubation, the cell was collected, washed,whereby preparing a washed cell.

On the other hand, washed cells of JM109/pUC413H and JM109/pUC413C wereprepared similarly to Example 3, added each at 1 g/dl, together with thewashed cell of the hydantoin racemase-expressing strain described above,to 0.1 mM KPB (pH 7.5) containing 1 g/dl of DL-5-benzylhydantoin andreacted at 30° C. By sampling at 24, 48 and 72 hours after the reactionfollowed by centrifugation followed by analysis of the supernatant byHPLC, D-phenylalanine produced was quantified.

The results are shown in Table 5. As evident from this table, by usingthe washed E.coli cells in which the hydantoin racemase gene,D-hydantoinase gene and D-carbamylase gene were expressed,D-phenylalanine was produced efficiently from benzylhydantoine in the DLform.

TABLE 5 D-Phenylalanine production when combined with racemization byhydantoin racemase Hydantoin racemase D-Hydantoinase D-CarbamylasePhenylalanine gene-expressing gene-expressing gene-expressing productionstrain strain strain level (g/dl) JM109/pUCFHR JM109/pUC413HJM109/pUC413C 0.83 JM109/pUC18 JM109/pUC18 JM109/pUC18 0.00

EXAMPLE 6

A reaction was performed similarly to Example 5 to obtain 500 ml of thereaction solution containing D-phenylalanine. This reaction solution wascentrifuged (10,000G×10 minutes) to separate the cells, and thesupernatant was concentrated under reduced pressure to 20 ml, wherebyprecipitating a crystal of D-phenylalanine. The precipitated crystal wasrecovered by filtration through a paper filter to obtain a crudecrystal. The crude crystal (2.4 g) was dissolved by combining with 10 mlof water and 1 ml of concentrated sulfuric acid, to which 100 mg ofactivated carbon was added to decolorize the solution. Then, theactivated carbon was filtered off, and the filtrate was combined with 5ml of a 28% aqueous ammonia to adjust at pH 3.5, whereby precipitatingD-phenylalanine. Subsequently, the precipitated crystal was recovered byfiltration through a filter paper and dried to yield 1.8 g ofD-phenylalanine. An HPLC analysis revealed that the material purity was99% and the optical purity was 99% e.e. or higher.

According to the present invention, a D-hydantoinase gene andD-carbamylase gene can be expressed stably in a large amount in a hostcell such as a coliform microorganism. As a result, such an enzyme canreadily be prepared using such a transformant, resulting in an abilityof producing a D-amino acid useful in pharmaceuticals, chemicalindustrial products and food additives efficiently using such atransformant, extract therefrom as well as a purified enzyme and thelike.

Although the invention has been described with respect to a specificembodiment for a complete and clear disclosure, the appended claims arenot to be thus limited but are to be construed as embodying allmodifications and alternative constructions that may occur to oneskilled in the art which fairly fall within the basic teaching hereinset forth.

1. An isolated DNA having a base sequence represented by (a) or (b) andthat encodes a protein having D-hydantoinase activity, wherein (a) isthe base sequence represented by SEQ ID NO: 1 in the Sequence Listing;and (b) is a base sequence hybridizing with a complementary basesequence of the base sequence represented by SEQ ID NO: 1 in theSequence Listing at a salt concentration corresponding to 0.1×SSC, 0.1%SDS at 60°C.
 2. The DNA of claim 1, wherein the base sequence isrepresented by (a).
 3. The DNA of claim 1, wherein the base sequence isrepresented by (b).
 4. An isolated DNA encoding a protein havingD-hydantoinase activity and having an amino acid sequence represented by(c) or (d), wherein (c) is the amino acid sequence represented by SEQ IDNO: 2 in the Sequence Listing; (d) is an amino acid sequence resultingfrom the substitution, deletion, insertion, addition or inversion of oneto ten amino acid residues in the amino acid sequence represented by SEQID NO: 2 in the Sequence Listing.
 5. The DNA of claim 4, wherein theamino acid sequence is represented by (c).
 6. The DNA of claim 4,wherein the amino acid sequence is represented by (d).
 7. A recombinantDNA resulting from a connection of a DNA with a vector DNA, wherein theDNA has a base sequence represented by (a) or (b) and encodes a proteinhaving D-hydantoinase activity, wherein (a) is the base sequencerepresented by SEQ ID NO: 1 in the Sequence Listing; and (b) is a basesequence hybridizing with a complementary base sequence of the basesequence represented by SEQ ID NO: 1 in the Sequence Listing at a saltconcentration corresponding to 0.1×SSC, 0.1% SDS at 60° C.
 8. Therecombinant DNA of claim 7, wherein the vector DNA is derived from a pUCplasmid, pBR322 plasmid or a derivative thereof.
 9. The recombinant DNAof claim 7, wherein the base sequence is represented by (a).
 10. Therecombinant DNA of claim 7, wherein the base sequence is represented by(b).
 11. A recombinant DNA resulting from a connection of a DNA with avector DNA, wherein the DNA encodes a protein having D-hydantoinaseactivity and having an amino acid sequence represented by (c) or (d),wherein (c) is the amino acid sequence represented by SEQ ID NO: 2 inthe Sequence Listing; (d) is an amino acid sequence resulting from thesubstitution, deletion, insertion, addition or inversion of one to tenamino acid residues in the amino acid sequence represented by SEQ ID NO:2 in the Sequence Listing.
 12. The recombinant DNA of claim 11, whereinthe vector DNA is derived from a pUC plasmid, pBR322 plasmid or aderivative thereof.
 13. The recombinant DNA of claim 11, wherein theamino acid sequence is represented by (c).
 14. The recombinant DNA ofclaim 11, wherein the amino acid sequence is represented by (d).
 15. Acell transformed with a recombinant DNA resulting from a connection of aDNA with a vector DNA, wherein the DNA has a base sequence representedby (a) or (b) and encodes a protein having D-hydantoinase activity,wherein (a) is the base sequence represented by SEQ ID NO: 1 in theSequence Listing; and (b) is a base sequence hybridizing a complementarybase sequence of with the base sequence represented by SEQ ID NO: 1 inthe Sequence Listing at a salt concentration corresponding to 0.1×SSC,0.1% SDS at 60° C.
 16. The cell of claim 15, wherein the cell is derivedfrom Escherichia coli.
 17. The cell of claim 15, wherein the basesequence is represented by (a).
 18. The cell of claim 15, wherein thebase sequence is represented by (b).
 19. A cell transformed with arecombinant DNA resulting from a connection of a DNA with a vector DNA,wherein the DNA encodes a protein having D-hydantoinase activity andhaving an amino acid sequence represented by (c) or (d), wherein (c) isthe amino acid sequence represented by SEQ ID NO: 2 in the SequenceListing; (d) is an amino acid sequence resulting from the substitution,deletion, insertion, addition or inversion of one to ten amino acidresidues in the amino acid sequence represented by SEQ ID NO: 2 in theSequence Listing.
 20. The cell of claim 19 wherein the cell is derivedfrom Escherichia coli.
 21. The cell of claim 19, wherein the amino acidsequence is represented by (c).
 22. The cell of claim 19, wherein theamino acid sequence is represented by (d).
 23. A method for producing aprotein having D-hydantoinase activity, the method comprising:incubating a cell in a culture medium; and allowing a protein havingD-hydantoinase activity to be accumulated in one or both of the culturemedium and the cell, wherein the cell to be incubated being transformedwith a recombinant DNA resulting from a connection of a DNA with avector DNA, wherein the DNA has a base sequence represented by (a) or(b) and encodes a protein having D-hydantoinase activity, wherein (a) isthe base sequence represented by SEQ ID NO: 1 in the Sequence Listing;and (b) is a base sequence hybridizing with a complementary basesequence of the base sequence represented by SEQ ID NO: 1 in theSequence Listing at a salt concentration corresponding to 0.1×SSC, 0.1%SDS at 60° C.
 24. The method of claim 23, wherein the base sequence isrepresented by (a).
 25. The method of claim 23, wherein the basesequence is represented by (b).
 26. A method for producing a proteinhaving D-hydantoinase activity, the method comprising: incubating a cellin a culture medium; and allowing a protein having D-hydantoinaseactivity to be accumulated in one or both of the culture medium and thecell, wherein the cell to be incubated being transformed with arecombinant DNA resulting from a connection of a DNA with a vector DNA,wherein the DNA encodes a protein having D-hydantoinase activity andhaving an amino acid sequence represented by (c) or (d), wherein (c) isthe amino acid sequence represented by SEQ ID NO: 2 in the SequenceListing; (d) is an amino acid sequence resulting from the substitution,deletion, insertion, addition or inversion of one to ten amino acidresidues in the amino acid sequence represented by SEQ ID NO: 2 in theSequence Listing.
 27. The method of claim 26, wherein the amino acidsequence is represented by (c).
 28. The method of claim 26, wherein theamino acid sequence is represented by (d).
 29. A method for producingN-carbamyl-D-amino acid, the method comprising: producing a proteinhaving D-hydantoinase activity by incubating a cell in a culture medium;and allowing a protein having D-hydantoinase activity to be accumulatedin one or both of the culture medium and the cell; and producingN-carbamyl-D-amino acid by making the protein having D-hydantoinaseactivity react with a 5-substituted hydantoin, wherein the cell to beincubated being transformed with a recombinant DNA resulting from aconnection of a DNA with a vector DNA, wherein the DNA has a basesequence represented by (a) or (b) and encodes a protein havingD-hydantoinase activity, wherein (a) is the base sequence represented bySeq. ID No. 1 in the Sequence Listing; and (b) is a base sequencehybridizing with a complementary base sequence of the base sequencerepresented by Seq. ID No. 1 in the Sequence Listing at a saltconcentration corresponding to 0.1×SSC, 0.1% SDS at 60° C.
 30. Themethod of claim 29, wherein an enzyme racemizing a 5-substitutedhydantoin compound or a material containing the enzyme is allowed toreact with the 5-substituted hydantoin whereby racemizing the5-substituted hydantoin compound.
 31. The method of claim 29, whereinthe base sequence is represented by (a).
 32. The method of claim 29,wherein the base sequence is represented by (b).
 33. A method forproducing N-carbamyl-D-amino acid, the method comprising: producing aprotein having D-hydantoinase activity by incubating a cell in a culturemedium; and allowing a protein having D-hydantoinase activity to beaccumulated in one or both of the culture medium and the cell; andproducing N-carbamyl-D-amino acid by making the protein havingD-hydantoinase activity react with a 5-substituted hydantoin, whereinthe cell to be incubated being transformed with a recombinant DNAresulting from a connection of a DNA with a vector DNA, wherein the DNAencodes a protein having D-hydantoinase activity and having an aminoacid sequence represented by (c) or (d), wherein (c) is the amino acidsequence represented by SEQ ID NO: 2 in the Sequence Listing; (d) is anamino acid sequence resulting from the substitution, deletion,insertion, addition or inversion of one to ten amino acid residues inthe amino acid sequence represented by SEQ ID NO: 2 in the SequenceListing.
 34. The method of claim 33, wherein an enzyme racemizing a5-substituted hydantoin compound or a material containing the enzyme isallowed to react with the 5-substituted hydantoin whereby racemizing the5-substituted hydantoin compound.
 35. The method of claim 33, whereinthe amino acid sequence is represented by (c).
 36. The method of claim33, wherein the amino acid sequence is represented by (d).
 37. A methodfor producing D-amino acid comprising: producing a protein havingD-hydantoinase activity by incubating a cell in a culture medium; andallowing a protein having D-hydantoinase activity to be accumulated inone or both of the culture medium and the cell; and producing D-aminoacid by making the protein having D-hydantoinase activity and an enzymehydrolyzing an N-carbamyl-D-amino acid or a material containing theenzyme react with a 5-substituted hydantoin, wherein the cell to beincubated being transformed with a recombinant DNA resulting from aconnection of a DNA with a vector DNA, wherein the DNA has a basesequence represented by (a) or (b) and encodes a protein havingD-hydantoinase activity, wherein (a) is the base sequence represented bySeq. ID No. 1 in the Sequence Listing; and (b) is a base sequencehybridizing with a complementary base sequence of the base sequencerepresented by Seq. ID No. 1 in the Sequence Listing at a saltconcentration corresponding to 0.1×SSC, 0.1% SDS at 60° C.
 38. Themethod of claim 37, wherein an enzyme racemizing a 5-substitutedhydantoin compound or a material containing the enzyme is allowed toreact with the 5-substituted hydantoin whereby racemizing the5-substituted hydantoin compound.
 39. The method of claim 37, whereinthe base sequence is represented by (a).
 40. The method of claim 37,wherein the base sequence is represented by (b).
 41. A method forproducing D-amino acid comprising: producing a protein havingD-hydantoinase activity by incubating a cell in a culture medium; andallowing a protein having D-hydantoinase activity to be accumulated inone or both of the culture medium and the cell; and producing D-aminoacid by making the protein having D-hydantoinase activity and an enzymehydrolyzing an N-carbamyl-D-amino acid or a material containing theenzyme react with a 5-substituted hydantoin, wherein the cell to beincubated being transformed with a recombinant DNA resulting from aconnection of a DNA with a vector DNA, wherein the DNA encodes a proteinhaving D-hydantoinase activity and having an amino acid sequencerepresented by (c) or (d), wherein (c) is the amino acid sequencerepresented by SEQ ID NO: 2 in the Sequence Listing; (d) is an aminoacid sequence resulting from the substitution, deletion, insertion,addition or inversion of one to ten amino acid residues in the aminoacid sequence represented by SEQ ID NO: 2 in the Sequence Listing. 42.The method of claim 41, wherein an enzyme racemizing a 5-substitutedhydantoin compound or a material containing the enzyme is allowed toreact with the 5-substituted hydantoin whereby racemizing the5-substituted hydantoin compound.
 43. The method of claim 41, whereinthe amino acid sequence is represented by (c).
 44. The method of claim41, wherein the amino acid sequence is represented by (d).